Study On Enzymatic Production Process Of Two D-Amino Acids

As the basic unit of protein,Amino acids are widely found in nature.They are also one of the important nutrients for human consumption.There are two kinds of isomers,D-form and L-form amino acid,which play different physiological functions in living organisms.D-p-hydroxyphenylglycine has an important application in the synthesis of ?-lactam semi-synthetic antibiotics.Being an important organic chiral source,D-Valine is mainly used for chiral Drugs,chiral additives and so on.This article will explore the enzymatic production process of two D-amino acids and provide a theoretical basis for industrial production.In this paper,two hydantoin racemase engineering strains were constructed.First,a hydantoin racemase gene from Rhodococcus sp.R04 was cloned and it was identified as a new gene by sequence alignment in the NCBI website Gen Bank.Second,by streamlining the cost optimization process,the experimental results show that the specific activity of N-carbamyl-D-amino acid hydrolase?CAB?when using Corn steep liquor medium was 0.6722 times that of CAB when using LB?Anqi?medium and 1.1860 times that of CAB when using LB?Oxide?medium.The cost of the medium is reduced by 1.33 yuan/L and 4.97 yuan/L,respectively.All results indicated that adding appropriate amount of activated carbon during the extraction process can remove the color of the reaction solution,simplifying the extraction steps.The new process reduces the cost of the medium,the amount of acid and wastewater discharge,etc and provide a theoretical basis for industrial production of D-amino acids.The two kinds of engineering strains which include hydantoin racemase gene were added in the conversion process of DL-5'-isopropyl hydantoin to D-Valine.When the conversion rate reached 50%,respectively.The conversion rate obviously increased after a transient inhibition.With the addition ofp MAL-s-hyu2/BL?DE3?,the conversion speed was increased by 12.12%,and the yield was over 99.0%.As to p RSET-hyu1/BL?DE3?,The conversion speed was increased by 18.18%,and the yield was also over 99.0%.The result showed addition of hydantoin racemase engineering strain increased the conversion rate and had no significant effect on the yield of the product.During the research,it was also found that D-hydantoinase which was isolated by ourselves from Pseudomonas sp.YZ26 in the enzymatic reaction can not only catalyze hydantoin and its 5'-single substitute product to prepare D-amino acids,but also has imidase activity.A high-performance liquid chromatography?HPLC?method was established for the detection of 2,3-pyridinedicarboximide?PDI?,phthalimide?PI?and their enzymatic reaction products 3-carbamoyl-?-picolinic acid??-3CP?,phthalamic acid?PA?.The test conditions were Hypersil^TM GOLD C18?4.6 mm × 250 mm,5 ?m?,and the mobile phase was H₂O-acetonitrile?90:10 by volume,containing 0.1% by volume of trifluoroacetic acid?at a flow rate of 1 m L/min.The PDI and PI reaction detection wavelengths are 254 nm and 220 nm,respectively,and the column temperature is room temperature.When the PDI and PI reached a saturated concentration in an aqueous solution,the specific activity of the engineering strain p ET3a-hyd/BL21?DE3?was determined to be 0.61 U/(m L·10 OD_600nm)and 0.15 U/(m L·10 OD_600nm).This study provides a solid theoretical basis for the future preparation of complex semi-amide organics using biological methods.In summary,we succeeded in cloning a new hydantoin racemase gene form Rhodococcus sp.R04 and constructed its engineering strain which can express the soluble recombinant enzyme.Our research lays a solid theoretical foundation for the industrial production of D-amino acids.The cyclic imide hydrolase activity of the D-hydantoinase used in our study was also discovered.